Joo Han Kim

Differentiation of intervertebral notochordal cells through live automated cell imaging system in vitro

Study Design. We demonstrated the differentiation of notochordal cells by direct observation using a live automated cell imaging system. We also hypothesized that notochordal cells have characteristics of chondrocyte-like cells. Objective. To determine characteristics of notochordal cells by matrix protein expression and their differentiation using a live automated cell imager. Summary of Background Data. Although notochordal cells are critical to homeostasis of intervertebral disc, their fate has not been extensively studied and there is little evidence of notochordal cells as progenitors. Methods. Notochordal cells purified from rabbit nucleus pulposus were isolated after serial filtration. Notochordal cells in 3-dimensional culture were compared to chondrocyte-like cells by 35S sulfate incorporation into proteoglycan and reverse transcription polymerase chain reaction for gene expression(collagen II and aggrecan). Notochordal cells in 2-D culture were used for immunocytochemical staining (collagen II, aggrecan, and SOX9) and time-lapsed cell tracking study. Results. Notochordal cells were capable of proteoglycan production at a rate comparable to chondrocyte-like cells (108% ± 22.6% to chondrocyte-like cells) and expressed collagen II, aggrecan, and SOX9. In time-lapsed cell tracking analysis, notochordal cells were slower in population doubling time than chondrocyte-like cells and differentiated into 3 morphologically distinct cell types: vacuolated cells (area: 2392 ± 507.1 &mgr;m2, velocity: 0.09 ± 0.01 &mgr;m/min); giant cells (area: 12678 ± 1637.0 &mgr;m2, velocity: 0.08 ± 0.01 &mgr;m/min) which grew rapidly without cell division; polygonal cells (area: 3053 ± 751.2 &mgr;m2, 0.14 ± 0.01 &mgr;m/min) morphologically similar to typical differentiation type of chondrocyte-like cells (area: 2671 ± 235.6 &mgr;m2, 0.19 ± 0.01 &mgr;m/min). Rarely, notochordal cells formed clusters analogous to that observed in vivo. Conclusion. These studies demonstrate a chondrocyte phenotype of notochordal cells and are the first direct evidence of notochordal cell differentiation, suggesting that they may act as progenitor cells, which has the potential to lead to their use in novel approaches to regeneration of degenerative intervertebral disc.

Upregulation of VEGF and FGF2 in normal rat brain after experimental intraoperative radiation therapy

The expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF)2 in the irradiated brain was examined to test how a single high dose radiation, similar to that used for intraoperative radiation therapy given to the normal cerebrum, can affect the vascular endothelium. After a burr hole trephination in the rat skull, the cerebral hemisphere was exposed to a single 10 Gy dose of gamma rays, and the radiation effect was assessed at 1, 2, 4, 6, and 8 weeks after irradiation. His-tological changes, such as reactive gliosis, inflammation, vascular proliferation and necrosis, were correlated with the duration after irradiation. Significant VEGF and FGF2 expression in the 2- and 8-week were detected by enzyme-linked immunosorbent assay quantification in the radiation group. Immunohistochemical study for VEGF was done and the number of positive cells gradually increased over time, compared with the sham operation group. In conclusion, the radiation injuries consisted of radiation necrosis associated with the expression of VEGF and FGF2. These findings indicate that VEGF and FGF2 may play a role in the radiation injuries after intraoperative single high-dose irradiation.

Activated macrophage-like THP-1 cells modulate anulus fibrosus cell production of inflammatory mediators in response to cytokines.

Study Design. Anulus fibrosus (AF) cells obtained from patients undergoing surgery were cocultured with macrophage-like cells and production of inflammatory mediators was analyzed by quantitative assay. Objective. To investigate the role of macrophages in AF cell production of inflammatory mediators by cytokines stimulation. Summary of Background Data. Discogenic pain caused by anular disruption is an important cause of low back pain and recent studies show the presence of macrophages in symptomatic discs but not in normal and aging discs. We hypothesize that macrophages play a major role in development of symptomatic disc. Methods. Human AF cells were cocultured with phorbol myristate acetate stimulated macrophage-like THP-1 cells. The conditioned medium from cells cultured alone or in coculture was assayed for cytokines by Enzyme-linked immunosorbent assay and nitric oxide (NO) by the Greiss method. Using the same outcome measures, comparisons of cell response to cytokines were made among macrophage-like cells, naïve AF cells, and macrophage exposed AF cells. Results. Tumor necrosis factor (TNF)-&agr;, interleukin (IL)-8, IL-6, and NO (TNF-&agr;: 1.45 ± 0.29 ng/mL, IL-8: 97.02 ± 7.94 ng/mL, IL-6: 33.40 ± 3.55 ng/mL, NO: 8.42 ± 0.78 &mgr;mol/L) were secreted in much greater amounts by cells maintained in coculture compared to macrophages (TNF-&agr;: 0.78 ± 0.12 ng/mL, IL-8: 58.04 ± 4.44 ng/mL, IL-6: 0.14 ± 0.03 ng/mL, NO: 0.30 ± 0.08 &mgr;mol/L) or AF cells cultured alone. In addition, IL-6 secretion from AF cells in response to TNF-&agr; was up-regulated by coculture, however, IL-6 secretion in response to IL-1 &bgr; was downregulated in a dose-dependent manner. Coculture with macrophages also up-regulated AF cell secretion of IL-8 dose-dependently and downregulated NO to TNF-&agr; or IL-1&bgr; stimulation. Conclusion. We conclude that exposure to macrophages, as can be expected after anular injury, can result in enhanced response to local inflammation. Although changes were observed in all inflammatory mediators after macrophage exposure, the most significant change was observed in IL-6 and IL-8, implicating these mediators in development of symptomatic disc.

Complete Freund's adjuvant-induced intervertebral discitis as an animal model for discogenic low back pain.

BACKGROUND: Although numerous animal models for low back pain associated with intervertebral disk (IVD) degeneration have been proposed, insufficient data have been provided to make any conclusions regarding pain. Our aim in this study was to determine the reliability of complete Freund’s adjuvant (CFA) injection into the rat spine as an animal model representing human discogenic pain. METHODS: We studied IVD degenerative changes with pain development after a 10-&mgr;L CFA injection into the L5-6 IVD of adult rats using behavioral, histologic, and biochemical studies. Serial histologic changes were analyzed to detect degenerative changes. Expression of calcitonin gene-related peptide (CGRP), prostaglandin E (PGE), and inducible nitric oxide synthase (iNOS) were determined using immunohistochemistry or real-time polymerase chain reaction as support data for pain development. In addition, CGRP immunoreactivity (ir) at the IVD was considered indirect evidence of neural ingrowth into the IVD. RESULTS: There was a significant increase of the hindpaw withdrawal response in the CFA group until 7 wk postoperatively (P < 0.05). Histologic analyses revealed progressive degenerative changes of the disks without any damage in adjacent structures, including nerve roots. In the CGRP-ir staining study, the bilateral dorsal horns and IVD had positive ir after intradiscal CFA injection. CGRP mRNA expression was increased in the dorsal root ganglion (DRG) at 2 and 4 wk, whereas PGE and iNOS mRNAs were markedly increased at 2 wk. The increment of CGRP expression was higher in allodynic rats compared with nonallodynic rats. CONCLUSION: Intradiscal CFA injection led to chronic disk degeneration with allodynia, which was suggested by pain behavior and expression of pain-related mediators. The increment of CGRP, PGE, and iNOS also suggest pain-related signal processing between the IVD and the neural pathway in this animal model. This animal model may be useful for future research related to the pathophysiology and development of novel treatment for spine-related pain.

Annulus Fibrosus Cells Interact With Neuron-Like Cells to Modulate Production of Growth Factors and Cytokines in Symptomatic Disc Degeneration

Study Design. We hypothesized that AF/neuron interactions during annular injury were involved in neovascularization and nerve ingrowth, the pathologic hallmarks of symptomatic disc degeneration. Objective. To identify growth factors and inflammatory cytokines related to AF/neuron interactions using in vitro model. Summary of Background Data. Discogenic pain is the chronic intractable pain initiated by tears in the outer annulus fibrosus (AF); this is a unique structure with free nerve endings at outer one-third, located beside dorsal root ganglia. The relationship between AF and neuron cells in annular injury has not been extensively investigated. Methods. Human AF cells were cocultured with a retinoic acid (RA)-treated SH-SY5Y human neuroblastoma cell line (neuron-like cells). Conditioned media from cells cultured alone or in coculture were assayed for growth factors and inflammatory cytokines using enzyme-linked immunosorbent assays. The responses of the neuron-like cells, the AF cells, and the cocultured group to IL-1&bgr;/TNF-&agr; were compared using the same outcome measures. Results. RA-treated SH-SY5Y cells showed significant neurite outgrowth on the 7th day; this is a typical morphologic finding of neuron-like cells. Neuron-like cells produced vascular endothelial growth factor (VEGF) and IGF-1 under basal conditions and dose-dependently secreted small amounts of IL-8 in response to TNF-&agr;. Coculturing enhanced the secretion of VEGF, TGF-&bgr;1, and &bgr;-NGF, and suppressed the production of IGF-1. VEGF in the coculture group and the AF cells was downregulated by IL-1&bgr;/TNF-&agr; stimulation. IL-1&bgr;/TNF-&agr; stimulation enhanced the production of large amounts of IL-6 and IL-8 from AF cells; IL-1&bgr; produced a greater response than TNF-&agr;. The neuron-like cells did not produce detectable amounts of IL-6 or IL-8. Conclusion. These studies suggest that AF cells are involved in an inflammatory reaction and that the interactions between AF and neuron-like cells enhance the production of growth factors responsible for neovascularization and nerve ingrowth. AF injury has the potential to initiate neovascularization/nerve ingrowth and an inflammatory reaction through the interactions of AF and neural tissues.

Spontaneous spinal epidural hematoma: An urgent complication of adding clopidogrel to aspirin therapy

We report a 56-year-old patient who had been taking antihypertensive medication in combination with prophylactic aspirin for 19 years who was diagnosed with stable angina with significant coronary artery stenosis on angiography. He was treated with drug-eluting coronary stent placement. Clopidogrel was added to the previous treatment regimen after stent placement. He visited the emergency room with complaints of severe back pain accompanied by radiculopathy and left leg weakness. The patient had an excellent outcome after immediate diagnosis by MRI and emergent evacuation of spontaneous spinal epidural hematoma (SSEH). The present case is interesting because it is the first case in spine which corresponds to the findings of MATCH study that bleeding tendency would be raised by dual antiplatelet treatment (aspirin+clopidogrel). With the popularity of antiplatelet medications, physicians should be aware of this critical side effect and provide urgent treatment. Furthermore, we should be cautious when we prescribe clopidogrel in addition to aspirin because it could cause bleeding complications like SSEH.

Brain Magnetic Resolution Imaging to Diagnose Bing-Neel Syndrome

Radiologic findings of Bing-Neel syndrome, which is an extremely uncommon complication resulting from malignant lymphocyte infiltration into the central nervous system (CNS) in patients with Waldenströms macroglobulinemia (WM), have been infrequently reported due to extreme rarity of the case. A 75-year-old man with WM presented at a neurology clinic with progressive gait and memory disturbances, and dysarthria of 2 months duration. Cerebrospinal fluid and serum protein electrophoresis and immunofixation electrophoresis showed IgM kappa-type monoclonal gammopathy. Brain magnetic resonance imaging revealed multifocal, hyperintense lesions on T2 weighted-images. Brain diffusion-weighted imaging (DWI) demonstrated hyperintensities in cerebral and cerebellar lesions that appeared isointense on apparent diffusion coefficient maps, which were compatible with vasogenic edema. Although histologic analysis is a confirmative study to prove direct cell infiltration into the brain, brain MRI with DWI may be a good supportive study to diagnose Bing-Neel syndrome.

The angiogenic capacity from ligamentum flavum subsequent to inflammation: a critical component of the pathomechanism of hypertrophy.

Study Design. In vitro study about angiogenic potentiality of ligamentum flavum (LF) cells using coculture of human lumbar LF cells and activated macropage-like THP-1 cells. Objective. To test our hypothesis that activated LF, which was exposed to inflammation, induces angiogenesis, thus resulting in hypertrophy. Summary of Background Data. Inflammatory reactions after mechanical stress produce fibrosis and scarring of the LF that result in hypertrophy, a major pathological feature of spinal stenosis. This study evaluated the roles of LF cells in the pathomechanism of hypertrophy, focusing on angiogenesis. Methods. To determine their response to the inflammatory reaction, human LF cells were cocultured with phorbol myristate acetate-stimulated macrophage-like THP-1 cells. The conditioned media were assayed for tumor necrosis factor (TNF)-&agr;, interleukin (IL)-1&bgr;, IL-6, IL-8, vascular endothelial growth factor (VEGF), and transforming growth factor (TGF)-&bgr;1. Naïve and macrophage-exposed LF cells that responded to TNF-&agr;/IL-1&bgr; were compared using the same outcome measures. Hypertrophied LF tissue was stained by TGF-&bgr;1 primary antibody using immunohistochemical method. Results. Larger quantities of IL-6, IL-8, and VEGF were secreted by cocultured cells than by macrophages alone and LF cells alone combined. Prior macrophage exposure increased the secretion of IL-8 and VEGF in response to TNF-&agr;/IL-1&bgr; stimulation whereas IL-6 production was increased in response to IL-1&bgr;. The coculture appeared to increase TGF-&bgr;1 secretion but the level was lower than that for macrophage-like cells alone and LF cells alone combined. Conclusion. LF cells interact with macrophage-like cells to produce angiogenesis-related factors except TGF-&bgr;1. Activated LF cells that have been exposed to macrophage, can impact the inducement of angiogenesis-related factors, suggesting that fibrosis and scarring during inflammatory reaction is the major pathomechanism of LF hypertrophy.

Low Level Light Therapy Modulates Inflammatory Mediators Secreted by Human Annulus Fibrosus Cells during Intervertebral Disc Degeneration In Vitro

Intervertebral disc degeneration (IVD) is one of the important causes of low back pain and is associated with inflammation induced by interaction between macrophages and the human annulus fibrosus (AF) cells. Low‐level light therapy (LLLT) has been widely known to regulate inflammatory reaction. However, the effect of LLLT on macrophage‐mediated inflammation in the AF cells has not been studied till date. The aim of this study is to mimic the inflammatory microenvironment and to investigate the anti‐inflammatory effect of LLLT at a range of wavelengths (405, 532 and 650 nm) on the AF treated with macrophage‐like THP‐1 cells conditioned medium (MCM) containing proinflammatory cytokines and chemokines (interleukin‐1beta, tumor necrosis factor‐alpha, interleukin‐6 and 8). We observed that AF cells exposed to MCM secrete significantly higher concentrations of IL‐6, IL‐8, IL‐1β and TNF‐α. LLLT markedly inhibited secretion of IL‐6 at 405 nm in a time‐dependent manner. Level of IL‐8 was significantly decreased at all wavelengths in a time‐dependent manner. We showed that MCM can induce the inflammatory microenvironment in AF cells and LLLT selectively suppressed IL‐6 and 8 levels. The results indicate that LLLT is a potential method of IVD treatment and provide insights into further investigation of its anti‐inflammation effect on IVD.

Notochordal cells influence gene expression of inflammatory mediators of annulus fibrosus cells in proinflammatory cytokines stimulation.

OBJECTIVE Notochordal cells in the intervertebral disc interact with nucleus pulposus (NP) cells and support the maintenance of disc homeostasis by regulation of matrix production. However, the influence of notochordal cells has not been evaluated in the annulus fibrosus (AF), which is the primary pain generator in the disc. We hypothesized that the notochordal cell has the capacity to modulate inflammatory mediators secreted by AF cells secondary to stimulation. METHODS Notochordal and AF cells were isolated from adult New Zealand white rabbits. AF pellets were cultured with notochordal cell clusters or in notochordal cell-conditioned media (NCCM) for 24 or 48 hours with proinflammatory cytokines at varying concentrations. Gene expression in AF pellets were assayed for nitric oxide synthase (iNOS), cyclo-oxygenase (COX)-2, and interleukin (IL)-6 by real time reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS AF pellet in NCCM significantly decreased the iNOS and COX-2 messenger ribonucleic acid (mRNA) levels compared to AF pellets alone and AF pellets with notochordal cells (p < 0.05). AF pellet resulted in dose-dependent iNOS and COX-2 expression in response to IL-1beta, stimulation, demonstrating that 1 ng/ml for 24 hours yielded a maximal response. AF pellet in NCCM significantly decreased the expression of iNOS and COX-2 in response to 1ng/ml IL-1beta, stimulation at 24 hours (p < 0.05). There was no difference in IL-6 expression compared to AF pellets alone or AF pellets with notochordal cell clusters. CONCLUSION We conclude that soluble factors from notochordal cells mitigate the gene expression of inflammatory mediators in stimulated AF, as expected after annular injury, suggesting that notochordal cells could serve as a novel therapeutic approach in symptomatic disc development.